Biomass-hydrolyzing Enzymes Research Articles

Systematic identification of CAZymes and transcription factors in the hypercellulolytic fungus Penicillium funiculosum NCIM1228 involved in lignocellulosic biomass degradation

BackgroundPenicillium funiculosum NCIM1228 is a filamentous fungus that was identified in our laboratory to have high cellulolytic activity. Analysis of its secretome suggested that it responds to different carbon substrates by secreting specific enzymes capable of digesting those substrates. This phenomenon indicated the presence of a regulatory system guiding the expression of these hydrolyzing enzymes. Since transcription factors (TFs) are the key players in regulating the expression of enzymes, this study aimed first to identify the complete repertoire of Carbohydrate Active Enzymes (CAZymes) and TFs coded in its genome. The regulation of CAZymes was then analysed by studying the expression pattern of these CAZymes and TFs in different carbon substrates—Avicel (cellulosic substrate), wheat bran (WB; hemicellulosic substrate), Avicel + wheat bran, pre-treated wheat straw (a potential substrate for lignocellulosic ethanol), and glucose (control).ResultsThe P. funiculosum NCIM1228 genome was sequenced, and 10,739 genes were identified in its genome. These genes included a total of 298 CAZymes and 451 TF coding genes. A distinct expression pattern of the CAZymes was observed in different carbon substrates tested. Core cellulose hydrolyzing enzymes were highly expressed in the presence of Avicel, while pre-treated wheat straw and Avicel + wheat bran induced a mixture of CAZymes because of their heterogeneous nature. Wheat bran mainly induced hemicellulases, and the least number of CAZymes were expressed in glucose. TFs also exhibited distinct expression patterns in each of the carbon substrates. Though most of these TFs have not been functionally characterized before, homologs of NosA, Fcr1, and ATF21, which have been known to be involved in fruiting body development, protein secretion and stress response, were identified.ConclusionsOverall, the P. funiculosum NCIM1228 genome was sequenced, and the CAZymes and TFs present in its genome were annotated. The expression of the CAZymes and TFs in response to various polymeric sugars present in the lignocellulosic biomass was identified. This work thus provides a comprehensive mapping of transcription factors (TFs) involved in regulating the production of biomass hydrolyzing enzymes.

Comparison of the solid-state and submerged fermentation derived secretomes of hyper-cellulolytic Penicillium janthinellum NCIM 1366 reveals the changes responsible for differences in hydrolytic performance

Solid-state fermentation (SSF) and submerged fermentation (SmF) have often been compared for production of biomass hydrolyzing enzymes highlighting the superiority of the SSF produced enzymes, but the reasons for the performance differences are under-explored. Penicillium janthinellum NCIM 1366 culture extracts from SSF had better hydrolytic performance along with a higher initial rate of reaction. Secretome analyses of the SSF and SmF enzymes using LC/MS-MS, indicated that while the type of proteins secreted were similar in both modes, the abundance of specific beta glucosidases, lytic polysaccharide monooxygenases and hemicellulolytic enzymes were very high in SSF resulting in efficient initiation, low accumulation of cellobiose and high initial reaction rates. Key enzymes that catalyse lignocellulose breakdown under SSF and SmF are therefore different and the fungus may be speculated to have regulation mechanisms that aid differential expression under different cultivation modes.

Advances and Challenges in Biocatalysts Application for High Solid-Loading of Biomass for 2nd Generation Bio-Ethanol Production

Growth in population and thereby increased industrialization to meet its requirement, has elevated significantly the demand for energy resources. Depletion of fossil fuel and environmental sustainability issues encouraged the exploration of alternative renewable eco-friendly fuel resources. Among major alternative fuels, bio-ethanol produced from lignocellulosic biomass is the most popular one. Lignocellulosic biomass is the most abundant renewable resource which is ubiquitous on our planet. All the plant biomass is lignocellulosic which is composed of cellulose, hemicellulose and lignin, intricately linked to each other. Filamentous fungi are known to secrete a plethora of biomass hydrolyzing enzymes. Mostly these enzymes are inducible, hence the fungi secrete them economically which causes challenges in their hyperproduction. Biomass’s complicated structure also throws challenges for which pre-treatments of biomass are necessary to make the biomass amorphous to be accessible for the enzymes to act on it. The enzymatic hydrolysis of biomass is the most sustainable way for fermentable sugar generation to convert into ethanol. To have sufficient ethanol concentration in the broth for efficient distillation, high solid loading >20% of biomass is desirable and is the crux of the whole technology. High solid loading offers several benefits including a high concentration of sugars in broth, low equipment sizing, saving cost on infrastructure, etc. Along with the benefits, several challenges also emerged simultaneously, like issues of mass transfer, low reaction rate due to water constrains in, high inhibitor concentration, non-productive binding of enzyme lignin, etc. This article will give an insight into the challenges for cellulase action on cellulosic biomass at a high solid loading of biomass and its probable solutions.

Comprehensive Optimization of Culture Conditions for Production of Biomass-Hydrolyzing Enzymes of Trichoderma SG2 in Submerged and Solid-State Fermentation.

Lignocellulose biomass contain large macromolecules especially cellulose and hemicelluloses that can be converted to fuel and chemicals using microbial biocatalysts. This study presents comprehensive optimization of production of biomass-hydrolyzing enzymes (BHE) by a high β-glucosidase-producing Trichoderma SG2 for bioconversion of lignocellulose biomass. Overall, a mixture of paper powder and switchgrass was most suited for production of BHE in submerged fermentation (SmF). BHE production was significantly different for various organic and inorganic nitrogen sources. The combination of peptone, yeast extract, and ammonium sulfate resulted in the highest activities (Units/mL) of BHE: 9.85 ± 0.55 cellulase, 38.91 ± 0.31 xylanase, 21.19 ± 1.35 β-glucosidase, and 7.63 ± 0.31 β-xylosidase. Surfactants comparably enhanced BHE production. The highest cellulase activity (4.86 ± 0.55) was at 25°C, whereas 35°C supported the highest activities of xylanase, β-glucosidase, and β-xylosidase. A broad initial culture pH (4-7) supported BHE production. The Topt for cellulase and xylanase was 50°C. β-xylosidase and β-glucosidase were optimally active at 40 and 70°C, respectively; pH 5 resulted in highest cellulase, β-glucosidase, and β-xylosidase activities; and pH 6 resulted in highest xylanase activity. Response surface methodology (RSM) was used to optimize major medium ingredients. BHE activities were several orders of magnitude higher in solid-state fermentation (SSF) than in SmF. Therefore, SSF can be deployed for one-step production of complete mixture of Trichoderma SG2 BHE for bioconversion of biomass to saccharide feedstock.

Production of thermostable β-glucosidase and CMCase by Penicillium sp. LMI01 isolated from the Amazon region

Background: Cellulolytic enzymes of microbial origin have great industrial importance because of their wide application in various industrial sectors. Fungi are considered the most efficient producers of these enzymes. Bioprospecting survey to identify fungal sources of biomass-hydrolyzing enzymes from a high-diversity environment is an important approach to discover interesting strains for bioprocess uses. In this study, we evaluated the production of endoglucanase (CMCase) and β-glucosidase, enzymes from the lignocellulolytic complex, produced by a native fungus. Penicillium sp. LMI01 was isolated from decaying plant material in the Amazon region, and its performance was compared with that of the standard isolate Trichoderma reesei QM9414 under submerged fermentation conditions. Results: The effectiveness of LMI01 was similar to that of QM9414 in volumetric enzyme activity (U/mL); however, the specific enzyme activity (U/mg) of the former was higher, corresponding to 24.170 U/mg of CMCase and 1.345 U/mg of β-glucosidase. The enzymes produced by LMI01 had the following physicochemical properties: CMCase activity was optimal at pH 4.2 and the β-glucosidase activity was optimal at pH 6.0. Both CMCase and β-glucosidase had an optimum temperature at 60°C and were thermostable between 50 and 60°C. The electrophoretic profile of the proteins secreted by LMI01 indicated that this isolate produced at least two enzymes with CMCase activity, with approximate molecular masses of 50 and 35 kDa, and β-glucosidases with molecular masses between 70 and 100 kDa. Conclusions: The effectiveness and characteristics of these enzymes indicate that LMI01 can be an alternative for the hydrolysis of lignocellulosic materials and should be tested in commercial formulations.

Transporter engineering in biomass utilization by yeast.

Biomass resources are attractive carbon sources for bioproduction because of their sustainability. Many studies have been performed using biomass resources to produce sugars as carbon sources for cell factories. Expression of biomass hydrolyzing enzymes in cell factories is an important approach for constructing biomass-utilizing bioprocesses because external addition of these enzymes is expensive. In particular, yeasts have been extensively engineered to be cell factories that directly utilize biomass because of their manageable responses to many genetic engineering tools, such as gene expression, deletion and editing. Biomass utilizing bioprocesses have also been developed using these genetic engineering tools to construct metabolic pathways. However, sugar input and product output from these cells are critical factors for improving bioproduction along with biomass utilization and metabolic pathways. Transporters are key components for efficient input and output activities. In this review, we focus on transporter engineering in yeast to enhance bioproduction from biomass resources.

RETRACTED: A biorefinery-based approach for the production of ethanol from enzymatically hydrolysed cotton stalks

Cotton post-harvest residue/cotton stalk (CS) – a major agro-residue in south asian countries was evaluated as a feed stock for bioethanol production. The common thermochemical pretreatment strategies based on dilute acid and alkali and different combinations of biomass hydrolyzing enzymes were evaluated for saccharification of CS biomass. A hydrolytic efficiency of 80% was achieved for alkali treated biomass using cellulase supplemented with beta glucosidase. Recycling of undigested/residual biomass and/or enzyme supported same final sugar concentration as for fresh hydrolytic experiments. Fermentation was carried out using a novel, inhibitor-resistant strain of Saccharomyces cerevisiae where 76% of theoretical maximum efficiency was attained. Material balances were derived for the entire process from biomass pre-processing to hydrolysis.

Whole-genome metabolic model of Trichoderma reesei built by comparative reconstruction.

Background Trichoderma reesei is one of the main sources of biomass-hydrolyzing enzymes for the biotechnology industry. There is a need for improving its enzyme production efficiency. The use of metabolic modeling for the simulation and prediction of this organism’s metabolism is potentially a valuable tool for improving its capabilities. An accurate metabolic model is needed to perform metabolic modeling analysis.ResultsA whole-genome metabolic model of T. reesei has been reconstructed together with metabolic models of 55 related species using the metabolic model reconstruction algorithm CoReCo. The previously published CoReCo method has been improved to obtain better quality models. The main improvements are the creation of a unified database of reactions and compounds and the use of reaction directions as constraints in the gap-filling step of the algorithm. In addition, the biomass composition of T. reesei has been measured experimentally to build and include a specific biomass equation in the model.ConclusionsThe improvements presented in this work on the CoReCo pipeline for metabolic model reconstruction resulted in higher-quality metabolic models compared with previous versions. A metabolic model of T. reesei has been created and is publicly available in the BIOMODELS database. The model contains a biomass equation, reaction boundaries and uptake/export reactions which make it ready for simulation. To validate the model, we dem1onstrate that the model is able to predict biomass production accurately and no stoichiometrically infeasible yields are detected. The new T. reesei model is ready to be used for simulations of protein production processes.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0665-0) contains supplementary material, which is available to authorized users.

New perspective on glycoside hydrolase binding to lignin from pretreated corn stover.

BackgroundNon-specific binding of cellulases to lignin has been implicated as a major factor in the loss of cellulase activity during biomass conversion to sugars. It is believed that this binding may strongly impact process economics through loss of enzyme activities during hydrolysis and enzyme recycling scenarios. The current model suggests glycoside hydrolase activities are lost though non-specific/non-productive binding of carbohydrate-binding domains to lignin, limiting catalytic site access to the carbohydrate components of the cell wall.ResultsIn this study, we have compared component enzyme affinities of a commercial Trichoderma reesei cellulase formulation, Cellic CTec2, towards extracted corn stover lignin using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and p-nitrophenyl substrate activities to monitor component binding, activity loss, and total protein binding. Protein binding was strongly affected by pH and ionic strength. β-d-glucosidases and xylanases, which do not have carbohydrate-binding modules (CBMs) and are basic proteins, demonstrated the strongest binding at low ionic strength, suggesting that CBMs are not the dominant factor in enzyme adsorption to lignin. Despite strong adsorption to insoluble lignin, β-d-glucosidase and xylanase activities remained high, with process yields decreasing only 4–15 % depending on lignin concentration.ConclusionWe propose that specific enzyme adsorption to lignin from a mixture of biomass-hydrolyzing enzymes is a competitive affinity where β-d-glucosidases and xylanases can displace CBM interactions with lignin. Process parameters, such as temperature, pH, and salt concentration influence the individual enzymes’ affinity for lignin, and both hydrophobic and electrostatic interactions are responsible for this binding phenomenon. Moreover, our results suggest that concern regarding loss of critical cell wall degrading enzymes to lignin adsorption may be unwarranted when complex enzyme mixtures are used to digest biomass.

Data for iTRAQ secretomic analysis of Aspergillus fumigatus in response to different carbon sources.

Here, we provide data related to the research article entitled “Quantitative proteomics study of Aspergillus fumigatus secretome revealed deamidation of secretory enzymes” by Adav et al. (J. Proteomics (2015) [1]). Aspergillus sp. plays an important role in lignocellulosic biomass recycling. To explore biomass hydrolyzing enzymes of A. fumigatus, we profiled secretome under different carbon sources such as glucose, cellulose, xylan and starch by high throughput quantitative proteomics using isobaric tags for relative and absolute quantification (iTRAQ). The data presented here represents the detailed comparative abundances of diverse groups of biomass hydrolyzing enzymes including cellulases, hemicellulases, lignin degrading enzymes, and peptidases and proteases; and their post translational modification like deamidation.

Semi reflective biopolymer layers for the detection of biomass hydrolytic enzymatic activities

ABSTRACTStructural colors were obtained by the deposition of plant cell walls biopolymers films on reflective support. Multilayered xyloglucan(XG)/cellulose nanocrystals(CN) thin films were obtained by spin-assisted layer-by-layer assembly while arabinoxylan (AX) thin films were elaborated via the spin-coating of AX/melamine formaldehyde resin followed by a cross-linking step. The effects of aqueous solutions on the stability of the structural colors were evaluated. The films were subsequently used to detect cellulase and xylanase activities by the change in the colors due to the film degradation. This enzymatic assay method appeared to be about 150 more sensitive that a standard method. Moreover due its simplicity, the method could be used to detect other biomass-hydrolyzing enzymes and more generally for other heterocatalytic degradations of solid polymer layers.

The Effects of Wheat Bran Composition on the Production of Biomass-Hydrolyzing Enzymes by Penicillium decumbens

The effects of the starch, protein, and soluble oligosaccharides contents in wheat bran on the extracellular biomass-hydrolyzing enzymes activities released by Penicillium decumbens mycelia grown in batch fermentations have been examined. The results showed increased starch content correlated directly with an increase in released amylase activity but inversely with the levels of secreted cellulase and xylanase. High amounts of protein in wheat bran also reduced the activities of cellulase, xylanase and protease in the culture medium. The effects of the soluble and insoluble components of wheat bran and cello-oligosaccharides supplements on production of extracellular cellulase and xylanase were compared. The soluble cello-oligosaccharides compositions in wheat bran were proved to be one of the most significant factors for cellulase production. According to the results of this research, determining and regulating the composition of wheat bran used as a fermentation supplement may allow for improved induction of cellulase and xylanase production.